Effect of Single Amino Acid Replacements on the Folding and Stability of Dihydrofolate Reductase from Escherichia coli

Kathleen M. Perry, C. Robert Matthews, James J. Onuffer, Mitchell S. Gittelman, Jin Tan Chen, Kazunari Taira, Stephen J. Benkovic, Nancy A. Touchette, Cinda S. Herndon, Ruth J. Mayer, Elizabeth E. Howell, Joseph Kraut

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Abstract

The role of the secondary structure in the folding mechanism of dihydrofolate reductase from Escherichia coli was probed by studying the effects of amino acid replacements in two a helices and two strands of the central β sheet on the folding and stability. The effects on stability could be qualitatively understood in terms of the X-ray structure for the wild-type protein by invoking electrostatic, hydrophobic, or hydrogen-bonding interactions. Kinetic studies focused on the two slow reactions that are thought to reflect the unfolding/refolding of two stable native conformers to/from their respective folding intermediates [Touchette, N. A., Perry, K. M., & Matthews, C. R. (1986) Biochemistry 25, 5445–5452], Replacements at three different positions in helix aB selectively alter the relaxation time for unfolding while a single replacement in helix aC selectively alters the relaxation time for refolding. This behavior is characteristic of mutations that change the stability of the protein but do not affect the rate-limiting step. In striking contrast, replacements in strands βF and βG can affect both unfolding and refolding relaxation times. This behavior shows that these mutations alter the rate-limiting step in these native-to-intermediate folding reactions. It is proposed that the intermediates have an incorrectly formed β sheet whose maturation to the structure found in the native conformation is one of the slow steps in folding.

Original languageEnglish (US)
Pages (from-to)2674-2682
Number of pages9
JournalBiochemistry
Volume26
Issue number10
DOIs
StatePublished - Jan 1 1987

All Science Journal Classification (ASJC) codes

  • Biochemistry

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    Perry, K. M., Matthews, C. R., Onuffer, J. J., Gittelman, M. S., Chen, J. T., Taira, K., Benkovic, S. J., Touchette, N. A., Herndon, C. S., Mayer, R. J., Howell, E. E., & Kraut, J. (1987). Effect of Single Amino Acid Replacements on the Folding and Stability of Dihydrofolate Reductase from Escherichia coli. Biochemistry, 26(10), 2674-2682. https://doi.org/10.1021/bi00384a004